Agarose Gel Electrophoresis

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nificant amounts of heat. The heat that is produced by passing the electrical current through the gel can behot enough to denature the DNA so that it runs through the gel as single strands instead of double strandsor the heat may even melt the gel. The buffer we will use today is a 0.5X TBE solution containing0.045M Tris-borate and 0.001M EDTA. To make our 0.5X buffer we will dilute a 10X TBE Bufferstock solution. Do you remember the functions of Tris and EDTA?We will use the GIBCO/BRL Horizon 58 Horizontal Gel Electrophoresis Apparatus for our agaroseelectrophoresis today.I. Assembly for Gel CastingA. Assemble the gel unit (Fig. 2) on a level surface (check with bubble level) following the procedure outlinedbelow.B. Open the safety interlocking lid and insert the buffer tray in the tray support stand.C. Place the gel deck in the center of the buffer tray with the outermost, well visualization strip (redstripe) towards the left (negative) electrode.D. Slide the gel casting dams (wedges) down in the "V"-shape grooves of the buffer tray. Keep the flatedges of the wedges against the gel casting deck. Apply gentle pressure simultaneously to both wedgesto seat the sealing surfaces against the sides of the gel deck. Do not force the gel casting dams down,as this may displace the gel deck out of level.E. Insert the comb into the desired comb position slot, with the teeth in line with a red stripe. The combsfor this unit have 8-teeth that are 1 mm wide and 0.8 mm thick, 8-teeth that are 1 mm wide and 1.5 mmthick, or 14-teeth that are 0.5 mm wide and 0.8 mm thick.Figure 2Gel Apparatus
II. Preparing 1% Agarose for GelsA. Weigh out 0.2 g of agarose into a 125 ml flask.B. Add 25 ml of autoclaved ddH 2 O to the flask.C. Microwave the flask for 1-2 min. Make sure that the mixture does not boil over. Stop the microwaveevery 30 s and swirl the flask. Continue to heat until the agarose is completely dissolved.D. After the agarose has completely dissolved, measure the volume of the contents of flask using a graduatedcylinder.E. Bring the volume up to 19 mL with autoclaved ddH 2 O and return the contents to the 125 ml flask.F. Add 1 mL of 10X TBE and swirl the flask.F. Microwave if necessary for 15-20 s.III. Pouring the GelA. Pour the contents of the flask into the gel casting deck. Pour carefully so than not many air bubblesare created. Also make sure that the entire bottom of the casting deck is covered.B. Remove any air bubbles with a pipet.C. Allow the gel apparatus to stand undisturbed until the agarose gel solidifiesIV. Preparing the SamplesWe will mix our DNA with a loading buffer to prepare the sample for loading onto the agarose gel. Theloading buffer contains glycerol and two dyes, bromophenol blue and xylene cyanol (the recipe for a 5Xconcentration of this loading buffer is shown below in Table 2). We use a loading buffer for two reasons.First, the glycerol in the loading buffer helps to keep our DNA in the loading well. The teeth of the combthat are placed in the gel will form a series of wells or depressions in the gel when the comb is removed.These wells represent the spaces in the gel that will hold our sample when we load the gel. When we removethe comb, 0.5X TBE buffer will fill the wells. As we add our sample to the wells the volume of oursample displaces the buffer in the wells. Glycerol is denser than water, and consequently, the loadingbuffer very readily displaces the 0.5X TBE buffer in the wells. Since our samples of DNA are mixed withthe loading buffer they tend to stay in the wells instead of diffusing away. The second reason we use aloading buffer is that the two dyes that are in the loading buffer will help us track how the gel is running.When 0.5% to 1.4% agarose gels are used with 0.5X TBE, the bromophenol blue front runs at about thesame position in the gel as 300 bp dsDNA and the xylene cyanol front runs at about the same position inthe gel as 4,000bp dsDNA.
Page 4 and 5: Table 2. Recipe for 5X Loading Buff
Page 6: D. Turn on the illuminator. You sho

Agarose Gel Electrophoresis

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